A safe and effective vaccine for HIV is critically needed to combat the worldwide scourge of AIDS. While the correlates of immune protection have yet to be clearly defined, either for protective or therapeutic vaccines, it is widely believed that both CD4+ and CD8+ T cell as well as humoral immunity are important. How sufficiently broad, potent, and sustained responses can be elicited has yet to be determined, and this represents a critical gap in our understanding of how to generate an effective vaccine such that protective immunity can be achieved. Dendritic cells (DC) represent the interface between innate and adaptive immunity and are among the most potent and important components in generating adaptive immune responses. In the present funding of this grant, we focused on novel approaches to manipulating DC in order to exploit their potential role in anti-HIV immune responses. In addition to demonstrating that mRNA loaded DC could induce CD4+, CD8+, and antibody responses, we identified the mechanisms whereby RNA activates innate immune receptors and developed RNA that avoids their activation and yields superior translational efficiency. Our hypotheses are that delivery of antigen-encoding, non-immunogenic mRNA into DC offers a uniquely flexible and potent approach to immunization, that DC activation after RNA delivery can be controlled to generate the desired immune responses (e.g. helper, cytotoxic, suppressor, and systemic or mucosal humoral immunity), and that this approach can be exploited for the development of HIV vaccines. The aim of this proposal is to optimize antigen presentation by DC of mRNA-encoded antigen, develop an in vivo delivery of mRNA, and establish proof-of-concept evidence for efficacy in HIV. To do this we will: 1) perform an analysis of translation, stability, and immune activation of nucleoside-modified mRNA. 2) optimize loading and activation of DC for cellular immune responses. 3) administer the mRNA vaccines in vivo. And, 4) test the mRNA vaccines in a Vaccinia-HIV challenge model. In these studies, we will combine two powerful approaches;DC based vaccines with mRNA encoded antigen to develop an easily deliverable vaccine. mRNA is complexed with adjuvants and given to subjects resulting in local loading and activation of DC. We anticipate that this work will enhance our understanding of RNA immunity, translation, and DC immune function;enable us to differentially manipulate specific elements in the immune response;and, ultimately, allow us to exploit mRNA encoded antigen generated immune responses for potentially protective and therapeutic purposes. In addition to providing a foundation for the rational development of a novel approach to HIV vaccine development, it is our hope that these studies may also offer a new avenue for vaccine development in general. PUBLIC HEALTH RELEVANCE: This project continues our studies in the development of an mRNA based vaccine. As the HIV epidemic continues to spread, the only effective approaches are education and combination drug therapy. We hope to develop an effective vaccine that will either prevent infection or alter disease course if infection occurs.